Water-soluble drugs and methods for their production

ABSTRACT

The present invention relates to water-soluble drugs, compositions containing same, and, in particular, a water-soluble analogue of geldanamycin. This invention also relates to a method of producing water-soluble analogues of water-insoluble drugs through derivatization and conjugation with a polar moiety via a thiol ether bond with a heterobifunctional linking molecule.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application No.09/743,873, now U.S. Pat. No. 6,747,055, filed Apr. 18, 2001, which isthe national stage filing of PCT/US99/16199, filed Jul. 15, 1999, whichclaims the benefit of U.S. Provisional Patent Application No.60/093,284, filed Jul. 17, 1998.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to water-soluble drugs, in particularwater-soluble analogues of geldanamycin, and compositions comprising thesame. This invention also relates to a method of renderingwater-insoluble drugs soluble in water and a method of treating cancer.

BACKGROUND OF THE INVENTION

A common problem associated with drugs intended for parenteral, andespecially intravenous, administration has been the solubilization of aslightly soluble or water-insoluble active ingredient (Sweetana et al.,PDA J. Pharm. Sci. & Tech., 50, 330 (1995)). As a result, many drugs ofpotential benefit in cancer chemotherapy and other areas of therapeuticshave been abandoned. Methods have been developed whereby drugs can beenveloped in micelles and placed into aqueous solutions (Hawthorne etal., J. Neurooncol., 33, 53–58 (1997)). Likewise, cosolvents andcomplexing agents allow some drugs to be dissolved in water (Badwan etal., U.S. Pat. No. 5,646,131). The use of these reagents, however, canbe complex and have negative attributes due to the additional reagentrequired to dissolve the active ingredient (Sweetana et al. (1995),supra). Prodrugs also have been developed by attaching groups, such asphosphates and other conjugates, to increase their solubility andenhance their performance (Schacter et al., Cancer Chemother.Pharmacol., 34, S58 (1994); Kingston et al., U.S. Pat. No. 5,278,324).

One water-insoluble drug of potential beneficial use in cancer therapyis geldanamycin. The drug is an ansamycin isolated from the broth ofStreptomyces hygroscopicus var. geldanus (DeBoer et al., Antiobiot., 23,442 (1970)). It has been found to exert its antiproliferating andanticancer activities by binding with the heat shock protein 90 (Hsp90)chaperone and, in turn, altering the translocation properties of thetumor suppressor protein p53 (Stebbins et al., Cell, 239 (1997);Sepehrnia et al., J. Biol. Chem., 271, 15,084 (1996); Dasgupta et al.,Experimental Cell Research, 29, 237 (1997)). Despite its therapeuticpotential as an anticancer agent, initial studies indicate that thebioavailability of geldanamycin must be enhanced and the toxicityassociated with the natural product reduced before significant progresscan be made with respect to the anticancer use of geldanamycin. Chemicalmodifications of geldanamcyin could potentially provide analogs withimproved bioactivity and bioavailability. While derivatives ofgeldanamycin have been developed to enhance the cancer-fighting effectsof the drug, the low solubility of such derivatives have required theuse of emulsifying or suspending agents in order to obtain aqueoussolutions. This has tended to reduce the bioavailability of the drug,and has thereby affected its utility as an anticancer agent.

The present invention addresses these problems by providing a method ofproducing water-soluble analogues of water-insoluble drugs and, inparticular, by providing a water-soluble analogue of the anticancer druggeldanamycin. Due to its thiol ether linkage, the analogue is expectedto exhibit superior bioavailability and stability under physiologicalconditions.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a water-soluble compound of the formula

where A is a water-insoluble drug, B₁ and B₂ together are a spacermoiety, and X is a polar moiety. The invention further provides apharmaceutical composition comprising a pharmaceutically acceptablecarrier and the above-described compound. In addition, the presentinvention provides a method of treating cancer in a mammal. The methodcomprises administering to a mammal having cancer an effective amount ofthe above-described compound.

The present invention further provides a method of rendering soluble inwater a water-insoluble drug. The method comprises contacting awater-insoluble drug comprising a side-chain that can react with abifunctional linking molecule with a bifunctional linking moleculecomprising a maleimido functional group to obtain a first derivative ofthe water-insoluble drug comprising a side-chain that comprises amaleimido functional group. The method further comprises contacting thefirst derivative with a polar moiety comprising a thio group (X—SH) toobtain a water-soluble compound as described above.

The present invention still further provides a water-soluble compound ofthe formula

or a pharmaceutically acceptable salt thereof, wherein:

R₁ is an ionic moiety bound to the carbon at position 17 via a nitrogenatom,

R₂ is a halo or an —OR₈ when there is a single bond between R₂ and thecarbon at position 11, wherein R₈ is hydrogen, a C_(1–C) ₈ alkylamido, aC₁–C₈ alkyl, a C₂–C₈ alkenyl, a C₂–C₈ alkynyl, a C₁–C₈ hydroxyalkyl, aC₁–C₈ alkyl carbamoyl, a C₁–C₈ alkylcarbonyl, or an aralkyl, any ofwhich R₈ can be further substituted with one or more substituents, whichcan be the same or different, selected from the group consisting of anitro, a halo, an azido, a hydroxy, an amido and an amino group, or

R₂ is oxo (═O) or oximino (═NOH) when there is a double bond between R₂and the carbon at position 11,

R₃ is selected from the group consisting of hydrogen and a group of theformula

wherein R₅, R₆, and R₇ are each independently selected from the groupconsisting of hydrogen, a halo, an azido, a nitro, a C₁–C₈ alkyl, aC₁–C₈ alkoxy, an aryl, a cyano, and an NR₁₀R₁₁R₁₂, wherein R₁₀, R₁₁, andR₁₂ are each independently selected from the group consisting ofhydrogen and a C₁–C₃ alkyl,

R₄ is selected from the group consisting of hydrogen, a halo, a C₁–C₈alkylamino, and a C₁–C₈ dialkylamino, and the bond between the carbonsat positions 4 and 5 can be a single bond or a double bond.

Also provided by the present invention is a water-soluble compound ofthe formula

or a pharmaceutically acceptable salt thereof, wherein:

Y is a spacer group,

P is a polypeptide or a protein that selectively binds to the surface ofa mammalian cell,

R₂ is a halo or an —OR₈ when there is a single bond between R₂ and thecarbon at position 11, wherein R₈ is selected from the group consistingof hydrogen, a C₁–C₈ alkylamido, a C₁–C₈ alkyl, a C₂–C₈ alkenyl, a C₂–C₈alkynyl, a C₁–C₈ hydroxyalkyl, a C₁–C₈ alkyl carbamoyl, a C₁–C₈alkylcarbonyl, and an aralkyl, any of which R₈ groups can be furthersubstituted with one or more substituents, which can be the same ordifferent, selected from the group consisting of a nitro, a halo, anazido, a hydroxy, an amido and an amino group, or

R₂ is oxo (═O) or oximino (═NOH) when there is a double bond between R₂and the carbon at position 11,

R₃ is selected from the group consisting of hydrogen and a group of theformula

wherein R₅, R₆, and R₇ are each independently selected from the groupconsisting of hydrogen, a halo, an azido, a nitro, a C₁–C₈ alkyl, aC₁–C₈ alkoxy, an aryl, a cyano, and an NR₁₀R₁₁R₁₂, wherein R₁₀, R₁₁, andR₁₂ are each independently selected from the group consisting ofhydrogen and a C₁–C₃ alkyl,

R₄ is selected from the group consisting of hydrogen, a halo, a C₁–C₈alkylamino, and a C₁–C₈ dialkylamino, and

the bond between the carbons at positions 4 and 5 can be a single bondor a double bond.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a reaction scheme illustrative of the present inventive methodby which the water-insoluble geldanamycin derivative is renderedwater-soluble.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides water-soluble compounds, in particular, awater-soluble analogue of geldanamycin, compositions comprising suchwater-soluble compounds and a method of producing water-solubleanalogues of water-insoluble drugs. Also provided is a method of usingsuch compounds to treat cancer.

Water-Soluble Drugs

The present inventive water-soluble compound has the formula

or a pharmaceutically acceptable salt thereof, wherein A is awater-insoluble drug, B₁ and B₂, together, are a spacer moiety, and X isa polar moiety. B₂ can be any suitable group lending a distance of atleast one carbon atom, and preferably less than twenty carbon atoms(e.g., one to ten carbon atoms), between the water-insoluble drug andthe maleimido functional group. Preferably, B₂ is selected from thegroup consisting of a C₁–C₁₉ alkylamido, a C₁–C₁₉ alkyl, a C₂–C₁₉alkenyl, a C₂–C₁₉ alkynyl, a C₁–C₁₉ hydroxyalkyl, a C₁–C₁₉alkycarbamoyl, a C₁–C₁₉ alkylcarbonyl, and an aralkyl, any of which canbe further substituted with one or more substituents, which can be thesame or different, selected from the group consisting of a nitro, ahalo, an azido, a hydroxy, an amido and an amino group. As meant hereinand throughout this disclosure an “aralkyl” moiety is preferably aC₁–C₂₀ alkyl, and more preferably a C₁–C₈ alkyl, wherein an alkylhydrogen atom is replaced by an aryl as defined herein. Examples ofaralkyl radicals include benzyl, phenethyl, 1-phenylpropyl,2-phenylpropyl, 3-phenylpropyl, 1-naphthylpropyl, 2-naphthylpropyl,3-naphthylpropyl, 3-naphthylbutyl, and the like. The term “aryl” refersto an aromatic carbocyclic radical, as commonly understood in the art,and includes monocyclic and polycyclic aromatics such as, for example,phenyl and naphthyl radicals, which radicals are, unless indicatedotherwise, optionally substituted with one or more substituents, whichare the same or different, selected from the group consisting of ahalogen, an alkyl, an alkoxy, an amino, a cyano, a nitro, and the like.Preferably, the aryl moiety has one or more six-membered carbocyclicrings including, for example, one to three carbocyclic rings, such asphenyl, naphthyl, and biphenyl.

More preferably B₂ is selected from a group consisting of a C₁–C₇alkylamido, a C₁–C₇ alkyl, a C₂–C₇ alkenyl, a C₂–C₇ alkynyl, a C₁–C₇hydroxyalkyl, a C₁–C₇ alkylcarbamoyl, a C₁–C₇ alkylcarbonyl, or anaralkyl, wherein the aralkyl has one to three aryl ring structureshaving 5 or 6 ring atoms each, and the alkyl portion of the aralkylmoiety has one to eight carbon atoms, and any wherein any of theforegoing B₂ groups can be further substituted with one or moresubstituents, which can be the same or different, selected from thegroup consisting of a nitro, a halo, an azido, a hydroxy, an amido or anamino group.

B₁ can be a methylenyl, an amido, —N═, an amino, or a thiol maleimidogroup. B₁ is ordinarily derived from a suitable functional groupincorporated into a bifunctional (i.e., dimaleimido orheterobifunctional) linking molecule. Of course, the bifunctionallinking molecule can be one that is commercially available, such asthose available from Pierce, Rockford, Ill. Commercially availablebifunctional linking moieties tend to contribute a portion of thefunctional group to the molecules that form from their use in linkingreactions. Exemplary linking reactions giving rise to some of theseembodiments are depicted in the EXAMPLES section (below). A multiplicityof spacer groups can thereby be incorporated into the present inventivewater-soluble drug. One particular spacer group useful in the context ofthe present invention has the following structure:

X can be any group that exhibits polar characteristics, including, butnot limited to, the propensity to interact with other polar substancesthrough hydrogen-bonding forces, Van der Waals forces, or dipolemoments. X together with the remainder of the present inventivecompound, is such that the present inventive compound is water-soluble.For purposes of the present invention, X is preferably ionic, morepreferably zwitterionic at neutral pH. Preferably, ionic polar moietiesare charged (e.g., greater than about 50% charged) at neutral pH. Forzwitterionic polar moieties, it is preferable for the charges to bebalanced at a pH of about 4 to about 10. More preferably, thezwitterionic moiety has a zero net charge (i.e., balanced charges) at apH of about 6 to about 8. Additionally, the zwitterionic moietypreferably has at least about 0.8 negative charges and at least about0.8 positive charges. By way of example and for the purposes of thisinvention, NaCl in water contains 1.0 positive charge and 1.0 negativecharge.

Polypeptides, peptides, and amino acids tend to be polar, and frequentlyzwitterionic moieties and are useful in the context of the presentinvention. Proteins suitable for use in the context of the presentinvention comprise polypeptides incorporating amino acids that exist ina conformation associated with a biological function or structure thatis characteristic of a substantially similar molecule produced by aliving cell. Preferred amino acids useful in the context of the presentinvention include lysine and cysteine, in particular L-cysteine, becausethey contain reactive side-chain nitrogen and sulfur atoms,respectively, that react easily with the functional portions ofcommercially available linker molecules.

Any water-insoluble drug can be used in the context of the presentinvention. For the purposes of this invention, the term “drug” means anycompound which is biologically active, e.g., exhibits a therapeutic orprophylactic effect in vivo, or a biological effect in vitro. Forexample, the drug can be an antihypertension drug, an antibiotic drug,or an anticancer drug. The present invention is particularly useful forrendering macrolide and ansamacrolide drugs water-soluble, at least inpart because the efficacy of these drugs tends to be limited by theamount of the drug that can be administered without causing ananaphylactic-like response (sometimes called a “toxic manifestation” bythose skilled in the art in the context of cancer chemotherapy or theadministration of insoluble drugs). An anaphylactic-like response occurswhen a water-insoluble drug, or a drug that readily precipitates atpharmacoactive concentrations in a mammal's blood is administered atabove a minimum threshold rate or concentration. As is known in the art,an anaphylactic-like response is accompanied by severe toxicity,swelling at the site of administration, nausea and other seriousside-effects in a mammal. Geldanamycin, and geldanamycin derivatives,are particularly useful in conjunction with the present invention.Examples of geldanamycin derivatives that are useful in the context ofthe present invention are described elsewhere herein, and in U.S. Pat.No. 5,387,584 (to Schnur) and U.S. Pat. No. 4,261,989 (to Sasaki etal.), which also disclose methods for making geldanamycin derivatives.

The term “water-insoluble” as used herein means partially or completelyinsoluble in water, or partially or completely non-dispersible in water.A water-insoluble compound in the context of the present inventionpreferably has a solubility less than the minimum effectiveconcentration in physiological saline. In contrast, a “water-soluble”compound of the present invention preferably has a solubility equal to,or greater than, the minimum clinically-effective concentration inphysiological saline. A clinically-effective concentration of aderivative of an insoluble drug is a concentration that is less than theconcentration that will induce an anaphylaxis-like response in apatient, and equal to, or greater than, the minimum concentration atwhich a therapeutic effect can be observed. Preferably, the inventivewater-soluble compound is soluble to at least about 2 mM inphysiological saline, more preferably to at least about 6 mM inphysiological saline. A water-insoluble drug useful in the context ofthe present invention preferably has a solubility of less than about 2mM, and optionally has a solubility of less than about 0.02 mM, inphysiological saline. Of course, the skilled artisan will appreciatethat for any particular drug of interest, these concentrations can beempirically determined and can be higher or lower. Preferably, thepresent inventive water-soluble drug is at least 3% as active as thewater-insoluble drug from which it is obtained, and more preferably isat least 10% as active as the water-insoluble drug.

The present inventive compound can be in the form of a pharmaceuticallyacceptable salt. Suitable pharmaceutically acceptable acid additionsalts include those derived from mineral acids, such as hydrochloric,hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids,and organic acids, such as tartaric, acetic, citric, malic, lactic,fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids,for example p-toluenesulphonic acids.

Ionic Geldanamycin

The present invention also provides water-soluble derivatives ofgeldanamycin of the formula:

wherein R₁, R₂, R₃, and R₄ are defined below.

R₁ is an ionic moiety bound to the carbon at position 17 via a nitrogenatom. Preferably, the ionic moiety promotes solubility in water.Additionally, R₁ is preferably an aliphatic moiety that can, but neednot, comprise an aryl moiety and is substituted by one or more chargedmoieties. Preferred aliphatic moieties in the context of the presentinvention comprise organic molecules comprising less than about 200carbon atoms and biopolymers, as that term is commonly understood in theart, including, but not limited to, proteins, nucleic acids, andpolysaccharides. The charged moieties can be the same or different andcan be selected from the group consisting of carbamate, carbonate,carboxylate, phosphamate, phosphate, phosphonate, pyrophosphate,triphosphate, sulfamate, sulfate, sulfonate, a C₁–C₈ monoalkylamine thatis protonated at neutral pH, a C₁–C₄ dialkylamine that is protonated atneutral pH, and a C₁–C₄ trialkylammonium. The selection of R₁ ispreferably made such that it is charged at neutral pH (i.e., about pH7). Preferably, R₁ is selected from the group consisting of a C₁–C₁₉alkylamido, a C₁–C₁₉ alkyl, a C₂–C₁₉ alkenyl, a C₂–C₁₉ alkynyl, a C₁–C₁₉hydroxyalkyl, a C₁–C₁₉ alkyl carbamoyl, a C₁–C₁₉ alkylcarbonyl, and anaralkyl. More preferably, R₁ is selected from the group consisting of aC₁–C₇ alkylamido, a C₁–C₇ alkyl, a C₂–C₇ alkenyl, a C₂–C₇ alkynyl, aC₁–C₇ hydroxyalkyl, a C₁–C₇ alkyl carbamoyl, a C₁–C₇ alkylcarbonyl, anda monocarbocyclic aralkyl. Additionally, R₁ can comprise a nucleoside(including nucleotides), a saccharide (including disaccharides,trisaccharides, and, as suggested above, polysaccharides of 4 to about50 or 200 sugar residues). R₁ also can comprise an amino acid, inparticular a naturally occurring amino acid, such as one encoded by amammalian genome, in particular a human genome. Of these, lysine isamong the preferred amino acids because the epsilon-amino group candisplace the 17-methoxy group of geldanamycin to yield a solublederivative of geldanamycin. Where R₁ is an amino acid, suitable blockinggroups can be used to protect functional groups on the amino acid. Forexample, BOC can be used to protect the α-amino group of the amino acid(see, King et al., Bioconjugate Chem., 10, 279–88 (1999)). The “blocked”17-demethoxy-17-BOC-amino acid-geldanamycin can optionally be“unblocked” in accordance with methods well-known in the art.Additionally, it is preferable that R₁ be zwitterionic at neutral pH.Any of these R₁ moieties can be further substituted with one or moresubstituents, which can be the same or different, selected from thegroup consisting of a nitro, a halo, an azido, a hydroxy, an amido andan amino group.

R₂ can be a halo or —OR₈, in which case there is a single bond betweenR₂ and the carbon at position 11. R₈ is selected from the groupconsisting of hydrogen, a C₁–C₈ alkylamido, a C₁–C₈ alkyl, a C₂–C₈alkenyl, a C₂–C₈ alkynyl, a C₁–C₈ hydroxyalkyl, a C₁–C₈ alkyl carbamoyl,a C₁–C₈ alkylcarbonyl, and an aralkyl, wherein the alkyl portion of thearyl moiety preferably has one to eight carbon atoms. These R₈ groupscan be further substituted with nitro, halo, azido, hydroxy, amido oramino groups.

Alternatively, R₂ is oxo (═O) or oximino (═NOH), in which case R₂ isbonded to the carbon at position 11 via a double bond.

R₃ is selected from the group consisting of hydrogen and a group of theformula

wherein R₅, R₆, and R₇ are each independently selected from the groupconsisting of hydrogen, a halo, an azido, a nitro, a C₁–C₈ alkyl, aC₁–C₈ alkoxy, an aryl, a cyano, and an NR₁₀R₁₁R₁₂, wherein R₁₀, R₁₁, andR₁₂ are each independently selected from the group consisting ofhydrogen and C₁–C₃ alkyl.

R₄ is selected from the group consisting of hydrogen, a halo, a C₁–C₈alkylamino, and a C₁–C₈ dialkylamino, and the bond between the carbonsat positions 4 and 5 can be a single bond or a double bond or can bedihydrogenated.

In one particular embodiment of the present invention, the bond betweenthe carbons at positions 4 and 5 is a double bond, and R₂, R₃, and R₄are selected to correspond to the homologous groups in geldanamycin suchthat 17-R₁N-17-demethoxy-geldanamycin is obtained. Those skilled in theart will also appreciate that the present invention also comprises 18,21-dihydroquinones of the present invention. Moreover, embodimentswherein the water-soluble geldanamycin is at least 3% as effective, morepreferably at least 10% as effective, as geldanamycin at stopping theproliferation of N87 cells (a gastric carcinoma, from ATCC, Rockville,Md.) in vitro (when measured by the IC₅₀ for thymidine incorporation)are preferred. While not intending to be bound by any particular theory,it is believed that 17-demethoxy-17-aminoR₁ derivatives of geldanamycinare preferable to other derivatives of geldanamycin because they areeither pharmaco-active or readily converted to an active form in thecell.

Selectively Targeted Geldanamycin

The present invention also provides a water-soluble compound of theformula:

or a pharmaceutically acceptable salt thereof, wherein R₂, R₃, and R₄are as defined above, Y is a spacer group, and P is a polypeptide or aprotein that selectively binds to the surface of a mammalian cell.

Preferably, Y comprises a thio ether. While not intending to be bound byany particular theory, it is believed that thio ether linkages arestable in the blood of a mammal, whereas they are degraded byintracellular enzymes present in cells. One particular Y group useful inthe context of the present invention comprises

Preferably, this Y moiety comprising the maleimido thiol ether is bondedto P via a lysinyl residue of P. One suitable method for achieving anembodiment of the present invention comprising this Y moiety is depictedin FIG. 1, described below, and a specific embodiment is given inExample 1. This inventive method comprises exposing the protein to asuitable amount of Traut's reagent i.e.,

For each protein the amount of Traut's reagent is preferably determinedempirically, but can be based on the deductive calculations based onantibody reactions. When P is an antibody (i.e., a protein of about 150kDa), the molar ratio of Traut's reagent: Ab is at least about 1:1,preferably at least about 5:1, and is preferably less than about 30:1,more preferably less than 15:1. The thiolated protein is highly reactiveand should be reacted with a linking molecule as soon as possible. Thelinking molecule, in turn, is preferably bound to the insoluble drugbefore the P moiety is thiolated. The reaction of the thiolated proteinor polypeptide and the linking molecule is initiated, preferably lessthan 12 hours after completion of the traut reaction, more preferablyless than about 2 hours after the traut reaction. Optionally, thereaction and product can be maintained under inert gas, such as argon.

The reaction of the insoluble drug-linking molecule with theTraut's-derivatized protein is subject to statistical mechanics.Accordingly, any initial preparation (i.e., unpurified preparation) willhave a distribution of drug:protein ratios, wherein each molecularproduct will have a ratio of n:1, wherein n is an integer (unless theprotein exists in a complex), and wherein the population has an averageratio of n:m, wherein n and m can be any positive number and need not beintegers. However, it will be appreciated that too high or too low aratio will decrease drug-efficacy and can render the drug or proteincompletely inactive. Accordingly, the ratio of drug:protein ispreferably carefully controlled.

Preferably, the drug to protein ratio, especially when P is an antibody,is at least 0.1:1 (drug:protein), more preferably at least 0.5:1, andmore preferably at least 1:1. Additionally, the drug:protein ratioshould preferably be less than about 6:1, and more preferably less thanabout 3:1. Moreover, for smaller proteins and polypeptides of about 10kDa or less, these ratios are preferably decreased, such that the mostpreferred ratio is about 0.6 to about 1.4 (drug:protein).

In accordance with this inventive method, a preferred linking moietycomprising a 2-maleimido thiol ether with the structural formula

can be made.

Optionally, P can be a polypeptide or a protein that binds to anantigen. One suitable example of such a polypeptide or protein which isuseful in the context of the present invention is an antibody, or anantigenically reactive fragment thereof, which is optionally humanized.Examples of suitable antibodies include herceptin and e21. Herceptin isa monoclonal antibody that has been humanized according to methods knownin the art and which binds to, and is internalized by, cells expressingthe Her2 receptor. The antibody e21 (C. R. King, Georgetown University,Washington, D.C., U.S.A.) is also an antibody that binds to Her2 and isinternalized by cells expressing the Her2 receptor. The e21 antibody wasraised in mice challenged with a membrane preparation ofHer2-transfected mammalian cells in tissue culture. Equivalentantibodies can be raised according to standard methods known in the art.

Embodiments wherein P is an anti-Her2 antibody, or an antigenicallyreactive fragment thereof, are useful in the treatment of cancer,particularly breast cancer, ovarian cancer, lung cancer, and gastriccancer. Anti-Her2 antibodies per se, exhibit anti-proliferative effectson Her2-expressing cancer cells. In this regard, herceptin is currentlyapproved for clinical use in the therapeutic treatment of cancer and isexpected to be of particular utility in the treatment of metastaticbreast cancer. Surprisingly, when geldanamycin is linked through alinking moiety, preferably one containing a thiol ether linkage, theanti-proliferative effects against breast cancer cells, e.g., SKBr3cells (ATCC, Rockville, Md.), MDA-361/DYT2 (a subclone of the well-knownMDA-MB-361 cells which were selected for their ability to form tumors inathymic mice by repeated in vivo transfer), and N87 cells, is moreeffective at inhibiting the growth of the cancer cells than either ofthe antibody or geldanamycin (used at comparable concentrations) alone.Moreover, the toxicity of the selectively targeted geldanamycin issubstantially reduced in mammals because the conjugated geldanamycin issoluble and does not tend to induce an anaphylaxis-like response.Additionally, the adult T-cell leukemia (ATL) cell, HuT102, which is aHer2-negative cancer cell that is highly sensitive to unconjugatedgeldanamycin, is not sensitive to the selectively targeted geldanamycincompound of the present invention. Thus, the therapeutic index ofgeldanamycin and of anti-proliferative antibodies can be substantiallyincreased by conjugation of these moieties in accordance with thepresent invention. While not intending to be bound by any particulartheory, it is believed that the ability of e21, herceptin, and otherantibodies to be efficiently internalized by target cells substantiallyenhances the therapeutic effect of the present inventive selectivelytargeted geldanamycin. Preferably, the selectively targeted geldanamycinis internalized by a mammalian cell that has a receptor for P at leastfive times more efficiently than another mammalian cell, or an otherwiseidentical cell, that does not have a receptor for P. Preferably, theselectively targeted geldanamycin of the present invention isinternalized by a log phase-target cell in culture at least about 25% asrapidly as an e21:gekdanamycin conjugate of the present invention isinternalized into a log phase N87 cell grown in complete RPMI comprising10% fetal calf serum, glutamine and antibiotics.

Other P moieties useful in the context of the present invention areantibodies huB4, C225 (available from Imclone or John Mendlesohn,Memorial Sloan-Kettering, New York, N.Y.), BR96, and Zenapax. Theantibody huB4 (see, Chari et al., Cancer Research, 55, 4079–84 (1995);Stone et al., Blood, 88, 1188–97 (1996)) is a humanized anti-B4 antibodythat binds with high affinity to CD19 and is internalized by cells towhich it binds through CD19. The antibody C225 binds with high affinityto human epidermal growth factor receptor and is internalized by cellsto which it binds. C225 sensitizes bound cells to anticancer drugs, butthe selectively targeted geldanamycin of the present invention willinhibit the growth of cancer cells more effectively than cancer cellstreated with C225 and exposed to a pharmaceutically acceptableconcentration of water-insoluble geldanamycin. Br96 is a chimerichuman/mouse antibody that binds with high affinity to Lewis-Y antigenand is internalized by cells to which it is bound. Lewis-Y antigen isselectively overexpressed on human carcinoma cells (see, Tolcher, J.Clinical Oncology, 17, 478–484 (1999)). Any of these, or similar,antibodies can be P in the present inventive selectively targetedgeldanamycin.

In other embodiments of the present inventive selectively targetedgeldanamycin P can be a diabody, an Fab, an Fab′₂, a single-chainantibody, or a single-chain Fab. These antigen-binding proteins andpolypeptides can be made in accordance with methods well-known in theart. Moreover, any antigen-binding protein or polypeptide that is usefulin the context of the present invention optionally can be humanized,e.g., the complementarity determining regions of the antigen-bindingprotein or polypeptide can be preserved, while the remainder of theprotein can be replaced by suitable human sequences, in accordance withmethods known in the art. Additionally, the antigen-binding protein orpolypeptide can be cationized (see, Pardridge et al., J. Pharmacol. andExp. Therapeutics, 286, 548–54 (1998)) by converting carboxyl groups toextended primary amino groups. Additionally, Fv's and otherantigen-binding proteins or polypeptides of the present invention can bestabilized by treatment with disulfide (see, Reiter et al, J. Biol.Chem., 269, 18327 (1994)). Other suitable modifications of theantigen-binding protein are also known in the art.

Additionally, the moiety P of the present inventive selectively targetedgeldanamycin can be a non-antigen-binding protein that binds to amammalian cell and is preferably internalized by that cell. Preferably,the cell has a receptor specific for P that is overexpressed onpathogenic cells. Also preferably, the cell has a receptor for P whichis expressed only or mainly on pathogenic cells. For example, P can be asecreted protein or polypeptide, such as an interleukin. Interleukin-2is a one such suitable interleukin. Alternatively, P can be a growthfactor, such as insulin, insulin-like growth factor, tumor necrosisfactor, or epidermal growth factor. Other suitable embodiments of Pinclude heregulin (see, Yang et al., Clinical Cancer Research, 4,993–1004 (1998)) and vascular endothelial cell growth factor, itsisoforms, and processed forms (see, Olson et al., Int. J. Cancer, 73,865–70 (1997)).

Compositions

Any of the drug-containing compounds of the present invention can beincorporated into a pharmaceutical composition or used in a method oftreating cancer as described herein with respect to the presentinventive water-soluble drug.

Advantageously, these embodiments of the present invention increaseefficacy by increasing geldanamycin concentration in targeted cells andby decreasing the toxicity of the geldanamycin by increasing itssolubility. While not desiring to be bound by any particular theory, itis also believed that the toxicity of geldanamycin is reduced inselectively targeted embodiments of the present invention by selectivelytargeting geldanamycin to selected cells and by sterically blocking thegeldanamycin from acting on non-targeted cells by incorporating a bulkysubstituent at the 17-position of geldanamycin.

The present inventive composition, which is preferably a pharmaceuticalcomposition, comprises a carrier, preferably a pharmaceuticallyacceptable carrier, and a compound of the present invention. Thepharmaceutical composition can comprise more than one active ingredient,such as more than one compound of the present invention, or a compoundof the present invention in combination with another pharmaceuticallyactive agent or drug.

The carrier can be any suitable carrier. With respect to pharmaceuticalcompositions, the carrier can be any of those conventionally used and islimited only by chemico-physical considerations, such as solubility andlack of reactivity with the active compound(s), and by the route ofadministration. It will be appreciated by one of skill in the art that,in addition to the following described pharmaceutical composition, thecompounds of the present inventive methods can be formulated asinclusion complexes, such as cyclodextrin inclusion complexes, orliposomes.

The pharmaceutically acceptable carriers described herein, for example,vehicles, adjuvants, excipients, and diluents, are well-known to thosewho are skilled in the art and are readily available to the public. Itis preferred that the pharmaceutically acceptable carrier be one whichis chemically inert to the active compound(s) and one which has nodetrimental side effects or toxicity under the conditions of use.

The choice of excipient will be determined in part by the particularcompound, as well as by the particular method used to administer thecomposition. Accordingly, there is a variety of suitable formulations ofthe pharmaceutical composition of the present invention. The followingformulations for oral, aerosol, parenteral, subcutaneous, intravenous,intramuscular, interperitoneal, rectal, and vaginal administration areexemplary and are in no way limiting.

Injectable formulations are among those formulations that are preferredin accordance with the present inventive methods. The requirements foreffective pharmaceutical carriers for injectable compositions arewell-known to those of ordinary skill in the art (see, e.g.,Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company,Philadelphia, Pa., Banker and Chalmers, eds., pages 238–250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622–630(1986)). It is preferred that such injectable compositions beadministered intravenously, intratumorally (within the tumor), orperitumorally (near the outside of the tumor). It will be appreciated byone of skill in the art that various of the described injectablecompositions are suitable for intratumoral and peritumoraladministration.

Topical formulations are well-known to those of skill in the art. Suchformulations are particularly suitable in the context of the presentinvention for application to the skin.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or orange juice; (b) capsules, sachets,tablets, lozenges, and troches, each containing a predetermined amountof the active ingredient, as solids or granules; (c) powders; (d)suspensions in an appropriate liquid; and (e) suitable emulsions. Liquidformulations may include diluents, such as water and alcohols, forexample, ethanol, benzyl alcohol, and the polyethylene alcohols, eitherwith or without the addition of a pharmaceutically acceptablesurfactant. Capsule forms can be of the ordinary hard- or soft-shelledgelatin type containing, for example, surfactants, lubricants, and inertfillers, such as lactose, sucrose, calcium phosphate, and corn starch.Tablet forms can include one or more of lactose, sucrose, mannitol, cornstarch, potato starch, alginic acid, microcrystalline cellulose, acacia,gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium,talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid,and other excipients, colorants, diluents, buffering agents,disintegrating agents, moistening agents, preservatives, flavoringagents, and pharmacologically compatible excipients. Lozenge forms cancomprise the active ingredient in a flavor, usually sucrose and acaciaor tragacanth, as well as pastilles comprising the active ingredient inan inert base, such as gelatin and glycerin, or sucrose and acacia,emulsions, gels, and the like containing, in addition to the activeingredient, such excipients as are known in the art.

The present inventive compound, alone or in combination with othersuitable components, can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like. They also maybe formulated as pharmaceuticals for non-pressured preparations, such asin a nebulizer or an atomizer. Such spray formulations also may be usedto spray mucosa.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The present inventive compound can be administered in a physiologicallyacceptable diluent in a pharmaceutical carrier, such as a sterile liquidor mixture of liquids, including water, saline, aqueous dextrose andrelated sugar solutions, an alcohol, such as ethanol, isopropanol, orhexadecyl alcohol, glycols, such as propylene glycol or polyethyleneglycol, dimethylsulfoxide, glycerol ketals, such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such aspoly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester orglyceride, or an acetylated fatty acid glyceride with or without theaddition of a pharmaceutically acceptable surfactant, such as a soap ora detergent, suspending agent, such as pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral.

Suitable fatty acids for use in parenteral formulations include oleicacid, stearic acid, and isostearic acid. Ethyl oleate and isopropylmyristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-b-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations will typically contain from about 0.5 toabout 25% by weight of the active ingredient in solution. Preservativesand buffers may be used. In order to minimize or eliminate irritation atthe site of injection, such compositions may contain one or morenonionic surfactants having a hydrophile-lipophile balance (HLB) of fromabout 12 to about 17. The quantity of surfactant in such formulationswill typically range from about 5 to about 15% by weight. Suitablesurfactants include polyethylene sorbitan fatty acid esters, such assorbitan monooleate and the high molecular weight adducts of ethyleneoxide with a hydrophobic base, formed by the condensation of propyleneoxide with propylene glycol. The parenteral formulations can bepresented in unit-dose or multi-dose sealed containers, such as ampoulesand vials, and can be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid excipient, forexample, water, for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described.

Additionally, the present inventive compounds, or compositionscontaining those compounds, can be made into suppositories by mixingwith a variety of bases, such as emulsifying bases or water-solublebases. Formulations suitable for vaginal administration can be presentedas pessaries, tampons, creams, gels, pastes, foams, or spray formulascontaining, in addition to the active ingredient, such carriers as areknown in the art to be appropriate.

Method of Treating Cancer

The present inventive compound can be used for any suitable purpose. Forexample, the present inventive compound can be used for scientific andresearch purposes, such as in determining the types of cancer which canbe treated and the onset of which can be delayed or the progress ofwhich can be slowed by administration of the present inventivecompound(s).

The present inventive compound has particular usefulness in applicationsin vivo. For example, the present inventive compound can be used in theprevention, delay of onset, slowing of progress, or treatment of cancer.

The present inventive method of treating cancer in a mammal, which ispreferably a human, comprises administering to a mammal having cancer aneffective amount, i.e., an anticancer effective amount, of a compound ofthe present invention. A preferred compound for use in the presentinventive method of treating cancer is a compound comprising a proteinor a polypeptide covalently bonded to 17-demethoxy-17-amino-geldanamycinor a derivative thereof, particularly wherein the derivative comprises aprotein or polypeptide that binds to the surface of a cancer cell, orwherein the derivative is zwitterionic. Preferably, a protein orpolypeptide bonded to 17-demethoxy-17-amino-geldanamycin or a derivativethereof, is bonded via a bifunctional linking molecule comprising a thioether. Preferably, the protein or polypeptide binds to an antigen. Also,the compound is preferably internalized by the cell to which it isbound.

The method of treating cancer using the compound of the presentinvention can be made more effective by administering one or more otheranticancer compounds along with one or more other compounds of thepresent invention. These other anticancer compounds include, but are notlimited to, all of the known anticancer compounds approved for marketingin the United States and those that will become approved in the future.See, for example, Table 1 and Table 2 of Boyd, Current Therapy inOncology, Section I. Introduction to Cancer Therapy (J. E. Niederhuber,ed.), Chapter 2, by B.C. Decker, Inc., Philadelphia, 1993, pp. 11–22.More particularly, these other anticancer compounds include doxorubicin,bleomycin, vincristine, vinblastine, VP-16, VW-26, cisplatin,carboplatin, procarbazine, and taxol for solid tumors in general;alkylating agents, such as BCNU, CCNU, methyl-CCNU and DTIC, for brainor kidney cancers; and antimetabolites such as 5-FU and methotrexate forcolon cancer.

One skilled in the art will appreciate that suitable methods ofadministering compositions comprising the present inventive compound toan animal, such as a mammal, in particular a human, are available, and,although more than one route can be used to administer a particularcompound, a particular route can provide a more immediate and moreeffective reaction than another route. Accordingly, the herein-describedmethods are exemplary and are in no way limiting.

The dose administered to an animal, such as a mammal, in particular ahuman, should be sufficient to prevent cancer, delay its onset, or slow(or stop) its progression. One skilled in the art will recognize thatdosage will depend upon a variety of factors including the strength ofthe particular compound employed, as well as the age, species,condition, and body weight of the animal. The size of the dose will alsobe determined by the route, timing, and frequency of administration aswell as the existence, nature, and extent of any adverse side-effectsthat might accompany the administration of a particular compound and thedesired physiological effect.

Suitable doses and dosage regimens can be determined by conventionalrange-finding techniques known to those of ordinary skill in the art.Generally, treatment is initiated with smaller dosages, which are lessthan the optimum dose of the compound. Thereafter, the dosage isincreased by small increments until the optimum effect under thecircumstances is reached. The present inventive method will typicallyinvolve the administration of about 0.1 to about 100 mg of one or moreof the compounds described above per kg body weight.

Method of Producing a Water-Soluble Drug

The present inventive method of rendering soluble in water awater-insoluble drug comprises contacting a water-insoluble drugcomprising a side-chain that can react with a bifunctional linkingmolecule, such as one that comprises a maleimido functional group, toobtain a first derivative of the water-insoluble drug comprising areactive maleimido side chain. Then, by contacting the first derivativewith a polar moiety comprising a thio moiety (X-SH), a water-solublecompound of the formula

or a pharmaceutically acceptable salt thereof, is obtained, wherein A isthe water-insoluble drug, B₁ and B₂ together are a spacer moiety, and Xis a polar moiety. The water-insoluble drug, spacer moiety, and polarmoiety are as previously described.

The water-insoluble drug optionally can be first reacted with amodifying agent to provide the aforementioned side-chain on the drug.The modifying agent can be any suitable agent that can produce aside-chain on the water-insoluble drug that can react with abifunctional linking molecule. Preferably, the water-insoluble drugcomprises a reactive methoxyaryl moiety, e.g., a methoxyquinone, thatcan react with a modifying agent comprising a primary amine. Reaction ofthe water-insoluble drug with the modifying agent then provides ademethoxy derivative of the water-insoluble drug in which the side-chaincomprises a primary or secondary amine that can react with abifunctional linking molecule. One preferred modifying agent is adiaminoalkyl, e.g., a C₁–C₂₀ alkyl comprising an amine on the first andan ultimate carbon, and is more preferably 1,3-diaminopropane or1,4-diaminobutane

While any one suitable bifunctional linking molecule can be used inconjunction with the present invention as described above, the linkingmolecule optionally can be selected from the group consisting ofN-γ-maleimidobutyryloxy-succinimide ester (GMBS),sulfo-N-γ-maleimidobutyryloxysuccinimide ester (sulfo-GMBS),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS),succinimidyl-4-[p-maleimidophenyl]butyrate (SMPB),sulfosuccinimidyl-4-[p-maleimidophenyl]butyrate (sulfo-SMPB),succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC),sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate(sulfo-SMCC), 4-[N-maleimidomethyl]-cyclohexane-1-carboxylhydrazide-HCl(M2C2H), and 4-[4-maleimidophenyl]-butyric acid hydrazide-HCl (MPBH).Most preferably, the bifunctional linking molecule issulfo-N-γ-maleimidobutyryloxysuccinimide ester (sulfo-GMBS).

Method of Making a Water-Soluble Geldanamycin

Geldanamycin (1 of FIG. 1) comprises a 17-methoxy moiety that isreactive with a primary amine in an organic solvent. Accordingly, any17-methoxy geldanamycin or its derivative can be reacted with a primaryamine to give a geldanamycin analogue that is reactive with a polarmoiety or a functional group of a mono- or bi-functional molecule orlinking molecule. Example 2 depicts various reaction schemes that can beused by those skilled in the art to make the present inventivecompounds. FIG. 1 illustrates a reaction of 3-amino-n-propylamine withgeldanamycin. The 3-amino-N-propylamine can be replaced with3-sulfhydryl-n-propylamine to create a geldanamycin that is reactivewith succinimidyl functional groups, rather than the maleimidylfunctional group illustrated in FIG. 1. Alternatively, lysine, orpreferably α-amino blocked-lysine (which can optionally be de-blockedsubsequently), can be directly reacted with geldanamycin to make awater-soluble derivative of geldanamycin, wherein the lysinyl residue isthe polar moiety, and wherein the polar moiety is ionic or zwitterionic.Additionally, the solvent system used to contact the geldanamycin can bemodified to facilitate the reaction. For example, when lysine is theprimary amine and is contacted to geldanamycin, it is acceptable to usea 5:5:1 mixture of chloroform:methanol:water, and preferable to use a1:1 mixture of chloroform:methanol. Of course, suitable substitutionsfor chloroform and methanol are within the spirit and scope of thepresent invention.

Various variations within the spirit and the scope of the presentdisclosure will be readily apparent to those of skill in the art.Moreover, any suitable, and preferably anticancer-effective, derivativeof geldanamycin can be substituted for the geldanamycin. Suchderivatives are well-known in the art. For example, U.S. Pat. No.5,387,584 (to Schnur) and U.S. Pat. No. 4,261,989 (to Sasaki et al.)disclose geldanamycin derivatives and methods for making the same.

EXAMPLES

The following examples further illustrates the present invention but, ofcourse, should not be construed as limiting the scope of the claimedinvention in any way.

Example 1

This example illustrates the preparation of a water-soluble analogue ofa water-insoluble drug in accordance with the present invention.

Geldanamycin 1 (see FIG. 1 for compounds referred to herein by number)was reacted with diaminopropane in chloroform to yield a mixturecomprising 17-aminopropylaminogeldanamycin 2 by way of the followingreaction. Geldanamycin (0.500 g, 0.0008918 mol) was dissolved inchloroform (200 ml). Diaminopropane (0.074 ml, 0.0008918 mol) was addeddropwise to the reaction flask and stirred at room temperature. Thereaction was monitored by thin layer chromatography (TLC) at regularintervals for the formation of the product.

Subsequent reaction of compound 2 withsulfo-N-g-maleimidobutyryloxysuccinimide ester (sulfo-GMBS) gave anintermediate 3 that could undergo Michael addition with compoundscontaining a thiol group. To accomplish this, a mixture of17-aminopropylaminogeldanamycin 2 (0.1000 g, 0.000166 mol) andsulfo-GMBS (0.0951 g, 0.0002489 mol) were stirred in chloroform at roomtemperature. The reaction mixture was partitioned between chloroform(200 ml) and water (100 ml). The chloroform fraction was separated,dried with sodium sulfate, and concentrated to dryness to give17-GMB-aminopropylaminogeldanamycin 3.

Compound 3 was reacted with L-cysteine to give the final product17-cys-GMB-aminopropylaminogeldanamycin 4, which is water-soluble. Toachieve the final product, a mixture of compound 3 (0.0500 g, 0.0000651mol) and L-cysteine (0.0316 g, 0.00026 mol) was stirred indimethylformamide (DMF) (4 ml) at room temperature overnight. Thereaction was monitored on a silica TLC plate (10% MeOH/CH₂Cl₂) thatshowed the desired product to be a purple spot at the point of origin.The reaction mixture was concentrated by using ethanol to form anazeotrope with DMF to give the crude reaction mixture (0.1074 g).

The reaction mixture was purified on C18 solid-phase extraction (SPE)columns with water and methanol (MeOH). Twelve 6-ml C18 SPE columns wereconditioned with MeOH (12 ml for each column) and water (12 ml for eachcolumn). Then the sample was dissolved in water (12 ml) and applied tothe twelve SPE columns (1 ml solution for each column). Each of thecolumns was eluted with water (3 ml) and MeOH (6 ml). The combined MeOHfractions were concentrated to give the final product 4, which was foundto be pure by NMR and FAB-MS analyses.

The analyses of compounds 2 through 4 were carried out by NMR andFAB-MS. Since there was a change of polarity from compound 3 to compound4, it should be noted that compound 3 was analyzed in both CD₂Cl₂ andd₄-methanol for its comparison with compounds 2 and 4, respectively.Extensive 1D and 2D NMR analysis allowed the unequivocal assignment ofmost of the proton and carbon signals, except for carbons 29–32 in thefive-membered ring. This was due to the fact that the thiol ether atcarbon 30 was added from both sides of the plane of the ring, resultingin a diastereomeric pair. Therefore, carbons 24 through 34 showed twopeaks and added further complexity in the spectrum. Taking the NMR andFAB-MS data as a complementary set, the structure for compound 4 wasconfirmed.

Additionally, the present example was repeated wherein diaminobutane wassubstituted for diaminopropane. This substitution facilitated reactionkinetics, and accordingly, is preferred for considerations pertaining tothe efficiency of compound synthesis.

Thus, the present invention provides an exemplary reaction sequence thatconverts a water-insoluble compound (e.g., 1, geldanamycin) to awater-soluble compound e.g., 4, in four, or preferably three steps. Theskilled artisan will appreciate that similar embodiments of the presentinvention can be readily discerned from the teachings of this example.

Example 2

This example illustrates nine reactions by which the chemical reactionsset forth in Example 1 can be modified to arrive suitably at othercompounds of the present invention. The general conditions of thesereactions are known in the art and can be adapted to use in the contextof the present invention without undue experimentation.

Example 3

This example demonstrates that suitable embodiments of the presentinventive incorporating geldanamycin have a higher therapeutic indexthan insoluble geldanamycin, because of a higher solubility and a lowertoxicity.

This example employs three antibodies, e21, AE1 (from Landolfi, ProteinDesign Labs, California), and anti-Tac (i.e., Zenapax fromHoffman-LaRoche, Inc., Nutley, N.J.). The antibodies e21 and AE1 bindHer2 with high affinity, and anti-Tac binds CD25 with high affinity. Allthree antibodies were radiolabeled and incubated with cells expressingthe respective ligands on their cell surfaces (N87 cells for e21 and AE1and HuT102 cells for anti-Tac). Both N87 cells and HuT102 cells arecancer cells that are known to be sensitive to the effects ofgeldanamycin. (HuT102 cells are cultured cells from an ATL patientavailable from the inventor's laboratories.) The cells were washed withdilute acid to remove unincorporated radiolabel, and the amount ofradiolabel remaining in the cells was measured as an indication of theamount of antibody internalized.

For e21, 10% of the radiolabel was taken up by N87 cells, while for AE1cells only 0% to 2% of radiolabel was taken up by N87 cells. Foranti-Tac, no significant quantity of radiolabel was taken up by HuT102cells. Accordingly, e21 is efficiently internalized by cells expressingHer2 on the cell surface, whereas AE1 and anti-Tac are not internalizedin significant quantities.

N87 cells were separately treated with e21, geldanamycin, and a presentinventive selectively targeted geldanamycin comprising e21 andgeldanamycin (“e21:geldanamycin conjugate”; per the method depicted inFIG. 1, except that the e21 antibody was treated with Traut's reagent togenerate free sulfhydryl groups). The e21 antibody alone did not have asubstantial effect on the proliferation of N87 cells, which was measuredby tritiated-thymidine incorporation (a standard method in the art).Geldanamycin inhibited 50% of the N87 proliferation at a concentrationof 8 nanomolar; 17-aminopropylamino-geldanamycin at 180 nanomolar. Incontrast, the e21:geldanamycin conjugate inhibited 50% of the N87proliferation at a concentration of about 300 nanomolar. Thus, bothgeldanamycin and the e21:geldanamycin conjugate effectively inhibit thegrowth of N87 cells, which express a receptor (Her2) for e21. However,in a clinical setting, unconjugated geldanamycin is toxicity-limited,due to its tendency to precipitate in a mammal's blood and to causeanaphylaxis and other serious side effects. Accordingly, conjugatede21:geldanamycin can be administered at a much higher concentration,which will be seen to give rise to a higher therapeutic index relativeto unconjugated geldanamycin.

In contrast, AE1 similarly conjugated to geldanamycin did not inhibitN87 proliferation by more than about 25%. Similarly, HuT102 cells, whichare sensitive to the effects of geldanamycin, were not substantiallyinhibited by an anti-Her2:geldanamycin conjugate made in accordance withthe method disclosed above. These data show that selectively targetedgeldanamycin conjugates have a markedly reduced effect on cells that donot bind to the conjugate. Accordingly, the toxicity to non-targetedcells is substantially reduced. This, of course, allows the skilledclinician to administer more of the drug to a mammal in need thereof,and further increases the therapeutic index of the present inventiveselectively targeted geldanamycin.

Example 4

This example demonstrates that 17-demethoxy-17-aminoderivatives ofgeldanamycin are effective inhibitors of cancer cell growth. N87 cellswere exposed to the 17-demethoxy-17-aminoderivative of geldanamycinindicated in Table 1 below, and the concentration at which theproliferation of the N87 cells was inhibited by 50% was determined innanomolar units.

TABLE 1 17-substituent IC50 (nM) OCH₃(geldanamycin) 8.4 NH(CH₂)₃NH₂ 180NH₂ 8.3 NHCH₂CH═CH₂ 5.7 NH(CH₂)₂Cl 0.6 NH(CH₂)₂OH 76 NH(CH₂)₂NH₂ Noteffective

All publications cited herein are hereby incorporated by reference tothe same extent as if each publication was individually and specificallyindicated to be incorporated by reference and was set forth in itsentirety herein.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truespirit and scope of the invention as defined by the claims herein.

1. A water-soluble compound of the formula

or a pharmaceutically acceptable salt thereof, wherein: R₁ is a chargedaliphatic moiety at neutral pH, which optionally is substituted by anaryl ring, wherein said aliphatic moiety is substituted by one or morecharged moieties, which can be the same or different, selected from thegroup consisting of carbamate, carbonate, phosphamate, phosphate,phosphonate, pyrophosphate, triphosphate, sulfamate, sulfate, a C₁–C₈monoalkylamine that is protonated at neutral pH, a C₁–C₄ dialkylaminethat is protonated at neutral pH, and a C₁–C₄ trialkylammonium, R₂ is ahalo or an —OR₈ when there is a single bond between R₂ and the carbon atposition 11, wherein R₈ is selected from the group consisting ofhydrogen, a C₁–C₈ alkylamido, a C₁–C₈ alkyl, a C₂–C₈ alkenyl, a C₂–C₈alkynyl, a C₁–C₈ hydroxyalkyl, a C₁–C₈ alkyl carbamoyl, a C₁–C₈alkylcarbonyl, and an aralkyl, any of which R₈ groups can be furthersubstituted with one or more substituents, which can be the same ordifferent, selected from the group consisting of nitro, halo, azido,hydroxy, amido and amino, or R₂ is oxo (═O) or oximino (═NOH) when thereis a double bond between R₂ and the carbon at position 11, R₃ isselected from the group consisting of hydrogen and a group of theformula

wherein R_(5,) R_(6,) and R₇ are each independently selected from thegroup consisting of hydrogen, a halo, an azido, a nitro, a C₁–C₈ alkyl,a C₁–C₈ alkoxy, an aryl, a cyano, and an NR₁₀R₁₁R_(12,) wherein R_(10,)R_(11,) and R₁₂ are each independently selected from the groupconsisting of hydrogen and a C₁–C₃ alkyl, R₄ is selected from the groupconsisting of hydrogen, a halo, a C₁–C₈ alkylamino, and a C₁–C₈dialkylamino, and the bond between the carbons at positions 4 and 5 canbe a single bond or a double bond.
 2. The compound of claim 1, whereinR₁ is substituted by a member selected from the group consisting of aC₁–C₁₉ alkylamido, a C₁–C₁₉ alkyl, a C₂–C₁₉ alkenyl, a C₂–C₁₉ alkynyl, aC₁–C₁₉ hydroxyalkyl, a C₁–C₁₉ alkyl carbamoyl, a C₁–C₁₉ alkylcarbonyl,and an aralkyl, any of which can be further substituted with one or moresubstituents, which can be the same or different, selected from thegroup consisting of a nitro, a halo, an azido, a hydroxy, an amido, andan amino group.
 3. The compound of claim 2, wherein R₁ is substituted bya member selected from the group consisting of a C₁–C₇ alkylamido, aC₁–C₇ alkyl, a C₂–C₇ alkenyl, a C₂–C₇ alkynyl, a C₁–C₇ hydroxyalkyl, aC₁–C₇ alkyl carbamoyl, a C₁–C₇ alkylcarbonyl, and a monocarbocyclicaralkyl any of which can be further substituted with one or moresubstituents, which can be the same or different, selected from thegroup consisting of a nitro, a halo, an azido, a hydroxy, an amido, andan amino group.
 4. The compound of claim 2, wherein said aliphaticmoiety comprises an amino acid.
 5. The compound of claim 1, wherein R₁is zwifferionic at neutral pH.
 6. A pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a compound ofclaim
 1. 7. A method of treating cancer in a mammal, wherein the canceris selected from the group consisting of endometrial carcinoma, breastcancer, leukemia, gastrointestinal cancer, a central nervous systemtumor and tongue carcinoma, wherein said method comprises administeringto a mammal having said cancer an effective amount of a composition ofclaim
 6. 8. A method of treating cancer in a mammal, wherein said methodcomprises administering to a mammal having cancer an anticancereffective amount of a composition of claim 6, whereupon the cancer inthe mammal is treated, and wherein the cancer is gastric carcinoma oradult T-cell leukemia.
 9. The method of claim 8, wherein the cancer isgastric carcinoma.
 10. The method of claim 8, wherein the cancer isadult T-cell leukemia.
 11. A water-soluble compound of the formula

or a pharmaceutically acceptable salt thereof, wherein: R₁ is a chargedaliphatic moiety at neutral pH, which optionally is substituted by anaryl ring, wherein said aliphatic moiety is substituted by one or morecharged moieties, which can be the same or different, selected from thegroup consisting of carbamate, carbonate, carboxylate, phosphamate,phosphate, phosphonate, pyrophosphate, triphosphate, sulfamate, sulfate,sulfonate, a C₁–C₈ monoalkylamine that is protonated at neutral pH, aC₁–C₄ dialkylamine that is protonated at neutral pH, and a C₁–C₄trialkylanimonium, R₂ is a halo or an —OR₈ when there is a single bondbetween R₂ and the carbon at position 11, wherein R₈ is selected fromthe group consisting of a C₁–C₈ alkylamido, a C₁–C₈ alkyl, a C₂–C₈alkenyl, a C₂–C₈ alkynyl, a C₁–C₈ hydroxyalkyl, a C_(1–C) ₈ alkylcarbamoyl, a C₁–C₈ alkylcarbonyl, and an aralkyl, any of which R₈ groupscan be further substituted with one or more substituents, which can bethe same or different, selected from the group consisting of nitro,halo, azido, hydroxy, amido and amino, or R₂ is oxo (═O) or oximino(═NOH) when there is a double bond between R₂ and the carbon at position11, R₃ is selected from the group consisting of hydrogen and a group ofthe formula

wherein R_(5,) R_(6,) and R₇ are each independently selected from thegroup consisting of hydrogen, a halo, an azido, a nitro, a C₁–C₈ alkyl,a C₁–C₈ alkoxy, an aryl, a cyano, and an NR₁₀R₁₁R_(12,) whereinR_(10, R) _(11,) and R₁₂ are each independently selected from the groupconsisting of hydrogen and a C₁–C₃ alkyl, R₄ is selected from the groupconsisting of hydrogen, a halo, a C₁–C₈ alkylamino, and a C₁–C₈dialkylamino, and the bond between the carbons at positions 4 and 5 canbe a single bond or a double bond.
 12. A water-soluble compound of theformula

or a pharmaceutically acceptable salt thereof, wherein: R₁ is a chargedaliphatic moiety at neutral pH, which optionally is substituted by anaryl ring, wherein said aliphatic moiety is substituted by one or morecharged moieties, which can be the same or different, selected from thegroup consisting of carbamate, carbonate, carboxylate, phosphamate,phosphate, phosphonate, pyrophosphate, triphosphate, sulfamate, sulfate,sulfonate, a C₁–C₈ monoalkylamine that is protonated at neutral pH, aC₁–C₄dialkylamine that is protonated at neutral pH, and a C₁–C₄trialkylanimonium, R₂ is a halo or an —OR₈ when there is a single bondbetween R₂ and the carbon at position 11, wherein R₈ is selected fromthe group consisting of hydrogen, a C₁–C₈ alkylamido, a C₁–C₈ alkyl, aC₂–C₈ alkenyl, a C₂–C₈ alkynyl, a C₁–C₈ hydroxyalkyl, a C₁–C₈ alkylcarbamoyl, a C₁–C₈ alkylcarbonyl, and an aralkyl, any of which R₈ groupscan be further substituted with one or more substituents, which can bethe same or different, selected from the group consisting of nitro,halo, azido, hydroxy, amido and amino, or R₂ is oxo (═O) or oximino(═NOH) when there is a double bond between R₂ and the carbon at position11, R₃ is selected from the group consisting of a group of the formula

wherein R_(5,) R_(6,) and R₇ are each independently selected from thegroup consisting of hydrogen, a halo, an azido, a nitro, a C₁–C₈ alkyl,a C₁–C₈ alkoxy, an aryl, a cyano, and an NR₁₀R₁₁R_(12,) wherein R_(10,)R_(11,) and R₁₂ are each independently selected from the groupconsisting of hydrogen and a C₁–C₃ alkyl, R₄ is selected from the groupconsisting of hydrogen, a halo, a C₁–C₈ alkylamino, and a C₁–C₈dialkylamino, and the bond between the carbons at positions 4 and 5 canbe a single bond or a double bond.
 13. A water-soluble compound of theformula

or a pharmaceutically acceptable salt thereof, wherein: R₁ is a chargedahiphatic moiety at neutral pH, which optionally is substituted by anaryl ring, wherein said aliphatic moiety is substituted by one or morecharged moieties, which can be the same or different, selected from thegroup consisting of carbamate, carbonate, carboxylate, phosphamate,phosphate, phosphonate, pyrophosphate, triphosphate, sulfamate, sulfate,sulfonate, a C₁–C₈ monoalkylamine that is protonated at neutral pH, aC₁–C₄ dialkylamine that is protonated at neutral pH, and a C₁–C₄trialkylanimonium, R₂ is a halo or an —OR₈ when there is a single bondbetween R₂ and the carbon at position 11, wherein R₈ is selected fromthe group consisting of hydrogen, a C₁–C₈alkylamido, a C₁–C₈ alkyl, aC₂–C₈ alkenyl, a C₂–C₈ alkynyl, a C₁–C₈ hydroxyalkyl, a C₁–C₈ alkylcarbamoyl, a C₁–C₈ alkylcarbonyl, and an aralkyl, any of which R₈ groupscan be further substituted with one or more substituents, which can bethe same or different, selected from the group consisting of nitro,halo, azido, hydroxy, amido and amino, or R₂ is oxo (═O) or oximino(═NOH) when there is a double bond between R₂ and the carbon at position11, R₃ is selected from the group consisting of hydrogen and a group ofthe formula

wherein R_(5,) R_(6,) and R₇ are each independently selected from thegroup consisting of hydrogen, a halo, an azido, a nitro, a C₁–C₈ alkyl,a C₁–C₈ alkoxy, an aryl, a cyano, and an NR₁₀R₁₁R_(12,) wherein R_(10,)R_(11,) and R₁₂ are each independently selected from the groupconsisting of hydrogen and a C₁–C₃ alkyl, R₄ is selected from the groupconsisting of a halo, a C₁–C₈ alkylamino, and a C₁–C₈ dialkylamino, andthe bond between the carbons at positions 4 and 5 can be a single bondor a double bond.
 14. The compound of claim 11, wherein R₁ issubstituted by a member selected from the group consisting of a C₁–C₁₉alkylamido, a C₁–C₁₉ alkyl, a C₂–C₁₉ alkenyl, a C₂–C₁₉ alkynyl, a C₁–C₁₉hydroxyalkyl, a C₁–C₁₉ alkyl carbamoyl, a C₁–C₁₉ alkylcarbonyl, and anaralkyl, any of which can be further substituted with one or moresubstituents, which can be the same or different, selected from thegroup consisting of a nitro, a halo, an azido, a hydroxy, an amido, andan amino group.
 15. The compound of claim 14, wherein R₁ is substitutedby a member selected from the group consisting of a C₁–C₇ alkylamido, aC₁–C₇ alkyl, a C₂–C₇ alkenyl, a C₂–C₇ alkynyl, a C₁–C₇ hydroxyalkyl, aC₁–C₇ alkyl carbamoyl, a C₁–C₇ alkylcarbonyl, and a monocarbocyclicaralkyl any of which can be further substituted with one or moresubstituents, which can be the same or different, selected from thegroup consisting of a nitro, a halo, an azido, a hydroxy, an amido, andan amino group.
 16. The compound of claim 14, wherein said aliphaticmoiety comprises an amino acid.
 17. The compound of claim 11, wherein R₁is zwitterionic at neutral pH.
 18. The compound of claim 12, wherein R₁is substituted by a member selected from the group consisting of aC₁–C₁₉ alkylamido, a C₁–C₁₉ alkyl, a C₂–C₁₉ alkenyl, a C₂–C₁₉ alkynyl, aC₁–C₁₉ hydroxyalkyl, a C₁–C₁₉ alkyl carbamoyl, a C₁–C₁₉ alkylcarbonyl,and an aralkyl, any of which can be further substituted with one or moresubstituents, which can be the same or different, selected from thegroup consisting of a nitro, a halo, an azido, a hydroxy, an amido, andan amino group.
 19. The compound of claim 18, wherein R₁ is substitutedby a member selected from the group consisting of a C₁–C₇ alkylamido, aC₁–C₇ alkyl, a C₂–C₇ alkenyl, a C₂–C₇ alkynyl, a C₁–C₇ hydroxyalkyl, aC₁–C₇ alkyl carbamoyl, a C₁–C₇ alkylcarbonyl, and a monocarbocyclicaralkyl any of which can be further substituted with one or moresubstituents, which can be the same or different, selected from thegroup consisting of a nitro, a halo, an azido, a hydroxy, an amido, andan amino group.
 20. The compound of claim 18, wherein said aliphaticmoiety comprises an amino acid.
 21. The compound of claim 12, wherein R₁is zwitterionic at neutral pH.
 22. The compound of claim 13, wherein R₁is substituted by a member selected from the group consisting of aC₁–C₁₉ alkylamido, a C₁–C₁₉ alkyl, a C₂–C₁₉ alkenyl, a C₂–C₁₉ alkynyl, aC₁–C₁₉ hydroxyalkyl, a C₁–C₁₉ alkyl carbamoyl, a C₁–C₁₉ alkylcarbonyl,and an aralkyl, any of which can be further substituted with one or moresubstituents, which can be the same or different, selected from thegroup consisting of a nitro, a halo, an azido, a hydroxy, an amido, andan amino group.
 23. The compound of claim 22, wherein R₁ is substitutedby a member selected from the group consisting of a C₁–C₇ alkylamido, aC₁–C₇ alkyl, a C₂–C₇ alkenyl, a C₂–C₇ alkynyl, a C₁–C₇ hydroxyalkyl, aC₁–C₇ alkyl carbamoyl, a C₁–C₇ alkylcarbonyl, and a monocarbocyclicaralkyl any of which can be further substituted with one or moresubstituents, which can be the same or different, selected from thegroup consisting of a nitro, a halo, an azido, a hydroxy, an amido, andan amino group.
 24. The compound of claim 22, wherein said aliphaticmoiety comprises an amino acid.
 25. The compound of claim 13, wherein R₁is zwitterionic at neutral pH.
 26. A pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a compound of claim11.
 27. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and a compound of claim
 12. 28. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and acompound of claim
 13. 29. A method of treating cancer in a mammal,wherein the cancer is selected from the group consisting of endometrialcarcinoma, breast cancer, leukemia, gastrointestinal cancer, a centralnervous system tumor and tongue carcinoma, wherein said method comprisesadministering to a mammal having said cancer an effective amount of acomposition of claim
 26. 30. A method of treating cancer in a mammal,wherein said method comprises administering to a mammal having cancer ananticancer effective amount of a composition of claim 26, whereupon thecancer in the mammal is treated, and wherein the cancer is gastriccarcinoma or adult T-cell leukemia.
 31. The method of claim 30, whereinthe cancer is gastric carcinoma.
 32. The method of claim 30, wherein thecancer is adult T-cell leukemia.
 33. A method of treating cancer in amammal, wherein the cancer is selected from the group consisting ofendometrial carcinoma, breast cancer, leukemia, gastrointestinal cancer,a central nervous system tumor and tongue carcinoma, wherein said methodcomprises administering to a mammal having said cancer an effectiveamount of a composition of claim
 27. 34. A method of treating cancer ina mammal, wherein said method comprises administering to a mammal havingcancer an anticancer effective amount of a composition of claim 27,whereupon the cancer in the mammal is treated, and wherein the cancer isgastric carcinoma or adult T-cell leukemia.
 35. The method of claim 34,wherein the cancer is gastric carcinoma.
 36. The method of claim 34,wherein the cancer is adult T-cell leukemia.
 37. A method of treatingcancer in a mammal, wherein the cancer is selected from the groupconsisting of endometrial carcinoma, breast cancer, leukemia,gastrointestinal cancer, a central nervous system tumor and tonguecarcinoma, wherein said method comprises administering to a mammalhaving said cancer an effective amount of a composition of claim
 28. 38.A method of treating cancer in a mammal, wherein said method comprisesadministering to a mammal having cancer an anticancer effective amountof a composition of claim 28, whereupon the cancer in the mammal istreated, and wherein the cancer is gastric carcinoma or adult T-cellleukemia.
 39. The method of claim 38, wherein the cancer is gastriccarcinoma.
 40. The method of claim 38, wherein the cancer is adultT-cell leukemia.